Lipids
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), L-α-phosphatidylcholine, hydrogenated (Soy) (HSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (sodium salt) (DOPG) were purchased from Lipoid (Germany). 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid) succinyl] (nickel salt) (DGS-NTA(Ni)) was purchased from Avanti Polar Lipids (USA). Cholesterol was purchased from Sigma-Aldrich (Israel). DOPE-PEG4-biotin and DOPE-cy5 were synthesized by reacting DOPE with NHS-PEG4-biotin or NHS-cy5 (BDL pharma, China).
Liposome preparation and absorbance measurements
Liposomes were prepared using the ethanol injection method. Lipids were weighed and dissolved in absolute ethanol and subsequently injected into calcium-free Dulbecco’s phosphate buffer saline (PBS; Sigma-Aldrich) preheated to 65 °C reaching a final lipid concentration of 50 mM. The liposomes were extruded five times using a high-pressure Lipex extruder (Northern Lipids, Canada) through 400, 200, and 100 nm polycarbonate-etched membrane (Whatman, Newton, MA, USA) at 65 °C.
For the absorbance measurements, liposomes were diluted 50-fold and measured in a UV-star 96-well microplate (Greiner bio-one, Germany) using the Infinite 200PRO multimode reader (TECAN, Switzerland) controlled with the i-control 1.10 software. Based on the absorbance data, a Lorentzian function was fitted for each of the phospholipids using the curve fitting toolbox in Matlab (version 2021b). The Lorentzian function Eq. (2) is:
$$L=a/({(x-b)}^{2+c})+d$$
(2)
Where the constants a, b, c, and d denote the Lorentzian amplitude, center, width, and offset respectively, and x denotes the Lorentzian variable – photon energy.
Synthetic cell preparation
Preparation of bacterial lysate for synthetic cell solutions
S30 bacterial lysate was prepared as described previously from BL21(DE3) E. coli transformed with the T7 polymerase expressing TargeTron vector pAR121959. For Gaussia luciferase expressing synthetic cells, DTT and β-mercaptoethanol were excluded from the S30 solution during the lysate preparation.
Preparation of lipids in mineral oil
POPC was lyophilized (FreeZone 2.5; Labconco, USA) overnight. POPC and cholesterol were dissolved separately in chloroform at a concentration of 50 mg ml−1 each. 50 µl of each solution was added to 500 µl mineral oil (Sigma-Aldrich) in a glass vial. The mixture was vortexed and then heated at 80 °C for one hour. The obtained lipid oil was stored at room temperature for up to 2 weeks.
Emulsion transfer method
Synthetic cell preparation using the emulsion transfer method was performed as previously described with several modifications59. The synthetic cells’ inner solution was either prepared according to the components lists in Supplementary Tables 2 and 3 or using the E. coli S30 extract system for circular DNA (Promega).
Shaking method
Synthetic cell preparation using the shaking method was performed as previously described by Gopfrich, et al.48 6 mM of 100 nm liposomes were prepared using the thin film hydration method. A thin lipid film of POPC, DOPG, DGS-NTA(Ni), DOPE-PEG4-biotin and DOPE-Cy5 at molar ratios of 73.5:20:5:1:0.5 was hydrated with PBS containing 10 mM MgCl2 and extruded as described in the liposomes’ preparation section. 100 μl of liposomes diluted to 2 mM were mixed with 200 μl of FC-40 oil (FL-0005-HP; Iolitec, Germany) with 10 mM of PFPE–carboxylic acid (Krytox; Costenoble, Germany) and 0.8% of fluorosurfactant (RAN Biotechnologies, USA) and incubated overnight. The bottom layer was then removed and 100 μl of PBS, followed by 100 μl of 1H,1H,2H,2H-Perfluoro-1-octanol (PFO; Sigma-Aldrich), were added on top of the remaining top layer. After 40 min of incubation, the top layer containing the synthetic cells was extracted.
Percentage of active synthetic cells
The percentage of active SCs was measured with imaging flow cytometry using the AMNIS ImageStream®X Mk II (Luminex Corporation, USA, controlled with the AMNIS Inspire software, version 200.1.681.0) and analyzed using the IDEAS software (version 6.2). SCs with or without a plasmid DNA expressing super folder GFP (and composed of an internal solution as described in Supplementary Table 2) were incubated for 1 h at 37 °C and diluted 10-fold in the synthetic cells’ outer solution (Supplementary Table 4) before their analysis with the ImageStream®X. SCs events were manually verified using images obtained from the brightfield channel, and their activity level was assessed by measuring their GFP fluorescence intensity using the 488 nm laser and the 505-560 nm emission channel.
UV exposure induced DNA damage
10 μg ml−1 of DNA (full sequence available in Supplementary Data 1) in a modified synthetic cell internal solution containing only sucrose, HEPES KOH pH = 8, magnesium acetate, potassium acetate, ammonium acetate and PEG 6000 (in the same final concentrations reported in Supplementary Table 2) was exposed for 20 min to 254 nm UV light (R-52 Grid lamp; UVP, USA) placed 10 cm above a 96-well plate before or after encapsulation in synthetic cells. DNA from the samples exposed to UV before and after encapsulation was extracted and PCR amplified using Phusion polymerase (Thermo-fisher, USA). The reaction products were then separated on a 1% agarose gel (uncropped and unprocessed image is available in the source data).
Luminescence assays
Luminescence was measured using the Infinite 200PRO multimode reader (TECAN) controlled with the i-control 1.10 software. The CFPS or synthetic cells reactions were mixed in a 1:1 ratio with native coelenterazine (Nanolight, USA) just prior to the measurement.
Luciferase comparisons
For comparison of light production in Rluc and Gluc CFPS reactions with the unmodified internal (reducing) conditions, reactions were incubated at 37 °C and 1200 rpm for 1 h. The reactions were measured after addition of a final concentration of 5 μM CTZ in a 384-well white microplate. For comparison of light production in Rluc synthetic cells and Gluc synthetic with the modified internal (oxidizing) conditions, synthetic cells were incubated at 37 °C for 1 h. 40-fold diluted synthetic cells and CTZ in a final concentration of 5 μM were mixed and measured in a 384-well white microplate.
Comparison to commercial transcription-translation system
Gluc light emission was compared in CFPS reactions and SCs composed of either the PURExpress system supplemented with disulfide bond enhancer (NEB, USA) prepared according to the manufacturer’s protocol or the self-prepared internal solution (described in Supplementary Table 3). For encapsulation in SC, the commercial internal solution was supplemented with 200 mM sucrose and PEG 6000 (3% w/w) to adjust the solution’s density and allow generation of vesicles. All reactions were incubated at 37 °C for 1 h. Before the luminescence measurement, CFPS reactions were diluted 1000-fold and synthetic cells solutions were diluted to reach equal absorbance of 0.1 at OD400 to ensure similar cell density. The diluted samples were measured after addition of a final concentration of 5 μM of CTZ in a 384-well white microplate.
Luciferase incubation temperature comparison
Gluc expressing synthetic cells were generated and split into tubes incubated at either 30 °C or 37 °C. Following 1 h of incubation, the samples were measured after addition of a final concentration of 5 μM of CTZ in a 384-well white microplate.
Luciferase production kinetics
Gluc expressing synthetic cells were incubated at 37 °C. At each time point, 15 μl of synthetic cells were taken and diluted 4-fold in PBS. The diluted sample was measured after addition of a final concentration of 2.5 μM of CTZ in a 384-well white microplate.
Luciferase kinetics and re-activation of light emission
Gluc-expressing synthetic cells were incubated at 37 °C for 1 h. For measuring the luciferase reaction kinetics, the synthetic cells were subsequently diluted 400-fold, mixed with a final concentration of 100 μM CTZ and measured in a 96-well white microplate. Luminescence was measured every 30 s for 15 min. For luminescence re-activation measurements, the synthetic cells were diluted 4-fold and mixed with 0.125 nmol of native CTZ in a 384-well white microplate. After measuring luminescence in one-minute intervals for 5 min, a second dose of 0.125 nmol of native CTZ was added to the same well and luminescence was measured again for 5 min.
Synthetic cell size and concentration measurements
Size analysis of synthetic cells was performed by light diffraction using the Mastersizer 3000 (Malvern Instrument, UK). The synthetic cell concentration was measured by imaging flow cytometry using the AMNIS ImageStream®X Mk II (Luminex Corporation, USA) and analyzed using the IDEAS software (version 6.2). Synthetic cells were diluted 10-fold in the synthetic cells’ outer solution (Supplementary Table 4) and counted based on the number of events verified as synthetic cells using the images from the brightfield channel. The synthetic cell concentration was calculated by dividing the number of verified synthetic cells by the total volume of sample analyzed.
Cryogenic scanning electron microscopy imaging
Cryogenic scanning electron microscopy (cryo-SEM) imaging was performed using Zeiss Ultra Plus high-resolution SEM, equipped with a Schottky field-emission gun and with a BalTec VCT100 cold-stage maintained below −145 °C. Specimens were imaged at low acceleration voltage of 1 kV, and working distances of 3–5 mm. Both the Everhart Thornley (“SE2”) and the in-the-column (“InLens”) secondary electron imaging detectors were used. The energy-selective backscattered (“ESB”) detector was used for elemental contrast between the organic and the aqueous phases. Low-dose imaging was applied to all specimens to minimize radiation damages.
Specimens were prepared by the drop plunging method, a 3 μL drop of solution was set on top of a special planchette maintaining its droplet shape and was manually plunged into liquid ethane, after which it was set on top of a specialized sample table. The frozen droplets were transferred into the BAF060 freeze fracture system, where they were fractured by a rapid stroke from a cooled knife, exposing the inner part of the drop. They were then transferred into the pre-cooled HR-SEM as described above. Ideally, imaging was performed as close as possible to the drop surface, where cooling rate should be maximal.
Western blot analysis
Synthetic cell samples after protein production were diluted 8-fold and analyzed using SDS-PAGE with a 12% acrylamide gel. The gel was blotted onto a nitrocellulose membrane and blocked with 5% nonfat milk powder in Tris‐buffered saline. Gaussia luciferase Polyclonal Antibody (PA1-181, Invitrogen, USA) diluted 1:3750 in Tris‐buffered saline with 0.5% Tween‐20 and 0.5% nonfat milk powder was incubated with the membrane overnight at 4 °C. After washing, the blots were incubated with horseradish peroxidase‐conjugated goat anti‐rabbit secondary antibody (ab6721, Abcam, UK) diluted to 1:20,000 and developed with Clarity Western ECL Blotting Substrate (BioRad, USA). The results were imaged using the Fusion FX6 imaging system (Vilber, France) controlled with the Evolution-capt software (version 18.02). Uncropped and unprocessed image is available in the source data.
For quantification of Gluc production, analysis of the images was performed with ImageJ gel analysis plug-in and use of a calibration curve for Gluc in known concentrations.
Photo-activation of conidiation in fungi with synthetic cells
Fungal growth
A Trichoderma atroviride inoculum was plated on the center of a PDYC (24 g l−1 potato dextrose broth (Difco, UK), 1.2 g l−1 casein hydrolysate (Sigma-Aldrich), 2 g l−1 yeast extract (Difco) agar plate and incubated for twenty-four hours in dark conditions. 3 ml of PDYC media were subsequently added to a new 10 cm culture plate. In the center of the plate a Whatman 50 filter paper cut to a diameter of 9 cm was placed over an 8 cm Whatman 1 filter paper. On top of these, a 0.5 cm square from the periphery of the fungal growth on the agar plate was placed in the center and incubated in the dark at room temperature for 48 h.
Exposure to synthetic cells
Synthetic cells were produced using the emulsion transfer method and diluted to the desired cell concentration. The synthetic cell concentration was quantified by flow cytometry using the AMNIS ImageStream®X Mk II as described above. Two consecutive exposures of the fungi to synthetic cells were performed. In each exposure, 500 μl of synthetic cells were mixed with 500 μl of native CTZ in a final concentration of 100 μM in a transparent plastic bag. The bag was localized over the periphery of the fungal colony in one part of the plate for 15 min and then removed. The plates were left for incubation in the dark for an additional period 24 h, after which they were imaged using a regular camera.
Image analysis
Background normalization of the images was performed manually in ImageJ (version 2.0.0) to achieve the same background average pixel value for all images60. The images were then converted to grayscale and then to black and white using a pixel threshold value equal to 45. The percentage of black pixels in a rectangle containing the top part of the plate where the synthetic cells were placed was calculated separately for each image, using python (version 3.7).
Generation of a light dose response curve
A dose response curve of T. atroviride sporulation was generated using a blue LED lamp (F-PLS10W/B 10 W, Eurolux). After growing the fungal colonies for 48 h in the dark, each colony was exposed to a specifically measured light dose. The total light dose ranged from 10 to 22500 μE m−2 as measured using a quantum radiometer photometer (Model LI-185B with a Quantum sensor, LI-COR, USA) by changing the exposure time of the colony (from 2 s to 15 min). After an incubation period of 24 h, the fungal colony-containing filter papers were transferred briefly to 70% ethanol and then fixed in absolute ethanol. Spores were isolated and suspended in 5 ml of water and their optical density was measured at 600 nm. One OD 600 unit corresponds to approximately 107 spores. A Michaelis-Menten curve was fitted using the least square regression method in Prism GraphPad. For analysis of the SC treatment, fungal colonies were treated as described in the “Exposure to synthetic cells” section above, followed by fixation and spore isolation to measure scatter.
Transcription activation in synthetic cells
LED illumination system
Five 470 nm blue LEDs (C503B-BAN-CY0C0461; Mouser, Israel) were connected together in series using an Arduino microcontroller and an external power supplier 0–30 V. On-off intervals were set by controlling relay modules using the Arduino IDE software. Blue light intensity was measured with a S310C light sensor (Thorlabs, USA).
EL222 mediated activation of transcription in cell free reactions
Cell-free reactions based on the internal synthetic cell solution (Supplementary Table 2) for Rluc expression or E. coli S30 extract system for circular DNA (Promega) for RFP expression were supplemented with 2.5 μM EL222 (unless stated otherwise) and incubated at 37 °C in 384-well microplates coated with an adhesive film to prevent evaporation. LEDs were placed 7 cm above the plate, providing approximately 12 W m−2, with on-off intervals of 20 and 70 s. For analysis of Rluc expression, native CTZ was added to a final concentration of 5 μM and luminescence was measured. For analysis of RFP expression, fluorescence intensity was measured with excitation / emission at 540 nm / 600 nm.
EL222 activation in synthetic cells
Synthetic cells with an internal solution based on the E. coli S30 extract system for circular DNA (Promega) were supplemented with 2.5 μM EL222. Prior to their incubation, each synthetic cell batch was divided into dark and light groups, both incubated at 37 °C in 384-well microplates coated with an adhesive film under dark or blue-light conditions. LEDs were placed 2.8 cm above the plate, providing approximately 19 W m−2, with on-off intervals of 20 and 70 s.
Self-activating synthetic cells based on the E. coli S30 extract system for circular DNA (Promega) were supplemented with 2.5 μM of Gluc-EL222. Prior to their incubation, each synthetic cell batch was divided into dark and light groups, both incubated at 37 °C. Native CTZ (0.2 nmol) was added every 30 min to the light group for a total of four times. The samples were further incubated for 90 more minutes after the last substrate addition. Just prior to the final fluorescence measurements, CTZ was added to the dark group in the same concentration that was added to the light group in order to eliminate differences due to substrate auto-fluorescence.
Membrane recruitment in synthetic cells
Membrane recruitment of RFP-sspB-Nano
A PDMS-walled chamber (Sylgard 184; Dow, USA), 5.5 mm in diameter and 2.5 mm height, was placed on a 22 × 50 mm deckglaser glass slide (Marienfeld, Germany). The bottom of the chamber was coated with 10 μg ml−1 streptavidin (Promega) overnight at 4 °C, or with 1% bovine serum albumin (Sigma-Aldrich) for 1 h at room temperature. The chamber was washed with PBS and in case of streptavidin coating, was coated once more for 1 h with 5 mg ml−1 of cold water fish skin gelatin (Sigma-Aldrich) and washed again with PBS.
Synthetic cells were prepared using the shaking method (see synthetic-cell preparation), and 100 nM of his-iLID or his-Gluc-iLID were added to the solution and incubated for 1 h at room temperature and shaking of 100 rpm. Under dark conditions, 25 nM of RFP-sspB-Nano were added to the cells and placed in the coated chamber in dark conditions for 30 min.
Imaging was performed using a standard inverted microscope (Eclipse Ti2; Nikon, controlled with NIS-Elements AR, version 5.11.01) outfitted with a 60×1.4 NA objective lens (Nikon). Low power laser illumination of approximately 5 mW cm−2 640 nm and 500 mW cm−2 561 nm lasers for imaging of Cy5 and mRFP1 respectively were used. Images were captured on a Sona sCMOS detector (Andor, Northern Ireland). Blue light laser illumination was performed with 488 nm laser at approximately 20 mW cm−2 with on-off intervals of 1.25 and 3.75 s for one minute. For recruitment in synthetic cells functionalized with his-Gluc-iLID, native coelenterazine (0.2 nmol) was added every minute for a total of four minutes.
Gluc-iLID light emission imaging
Synthetic cells functionalized with Gluc-iLID were imaged after addition of 100 μM substrate (final concentration) using a standard inverted microscope (Eclipse Ti2; Nikon) outfitted with a 40×0.75 NA objective lens (Nikon) and equipped with iXON EMCCD camera (Andor). Images were obtained with 400 msec exposure time and gain 300.
Statistics
The statistical analysis including student’s t-test, one-way, two-way ANOVA and Michaelis–Menten model fitting was performed using Prism GraphPad version 8.3.0.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
